Chemoproteomic Profiling of Cellular Substrates of the Lysine Acetyltransferase HAT1 Using Cell Permeable Bioorthogonal Reporters.

Although quantitative proteomics studies have identified thousands of acetylation substrates and acetylation sites in both eukaryotic and prokaryotic cells, our knowledge of the acetylation scopes contributed by individual lysine acetyltransferases (KATs) is still very limited. A representative example is that functional studies on the histone acetyltransferase 1 (HAT1), one of the first KAT members identified in mid-1990s, have been largely restricted on its nuclear histone substrate. Lacking information on the proteome-wide substrate profiles of HAT1 and other different KAT members greatly hinders our understanding of functional roles of this important family of enzymes in regulating cellular physiology and pathology. Recently, acyl-CoA reporters have emerged as effective tools to label KAT substrates in complex proteome settings, especially in whole cell lysates. However, it was commonly regarded that acyl-CoA molecules are not cell-permeable and cannot be applied for performing protein acylation profiling in living cells. Here, we report that a clickable acyl-CoA analog, 3-azidopropanoyl CoA (3AZ-CoA), showed remarkable cell permeability and effectively labeled proteins in cellulo. We rationally engineered the active site of HAT1 by site-directed mutagenesis and obtained a HAT1 mutant that showed excellent bioorthogonal activity utilizing 3AZ-CoA as a cofactor to effectively acylate and probe HAT1 substrates. Importantly, we were able to apply the bioorthogonal enzyme-cofactor pair to achieve HAT1 substrates labeling, enrichment, and identification from the living cells. The reactive azide warhead group successfully allowed for the labeled protein substrates to be pulled down following the chemoselective conjugation with the biotin handle through the copper-catalyzed alkyne-azide cycloaddition chemistry. Subsequent SILAC proteomic analysis of the bioorthogonally enriched proteins revealed hundreds of novel HAT1 substrates, which include proteins critical for transcription, RNA splicing, mitochondria, metabolism, and oxidation-reduction process pathways. These findings underline that HAT1 has broader functionalities in mammalian cells than previously thought. The methodology we applied herein also proves that cell-permeable acyl-CoA reporters that orthogonally match with corresponding KAT enzymes forms serve as a powerful chemical biology tool for functional mapping of the acetylomes of different KAT isoforms from their native biological contexts.